Chemotherapy, particularly with a combination of anti-cancer agents, is the treatment of choice for delocalized tumors that are untreatable by surgery or radiation. However, some patients relapse after even a short period of time, and do not respond to a second course of chemotherapy.
Most malignant tumors show some sensitivity to cytotoxic drugs. Accordingly, treatment with these drugs generally provides remission and shrinkage of the tumor that may last for weeks to months. Nonetheless, in many cases the tumors regrow, and this regrowth is resistant to further cytotoxic treatments.
Cancer chemotherapy has its roots in the antimicrobial chemotherapy that has been evolving since the beginning of the twentieth century. Since many microbes have now become resistant to classical antimicrobial chemotherapy, it is not surprising that clinical resistance to anticancer drugs is evident. Early experiments with tumors transplanted into mice demonstrated the development of progressive resistance to experimental drugs. Since those experiments, tumors resistant to every type of anticancer drug have been isolated. All organisms, including the cells within a malignant tumor, appear to have the ability to develop a resistance to drugs that would otherwise be fatal.
The underlying cause of progressive drug resistance, whether in infectious diseases or in cancer, is related to spontaneous genetic mutations which occur in all living cells, which mutations are inheritable and may be passed on to succeeding generations. In any cell population, mutants that are resistant to any given drug occur at a frequency of somewhere between one in 10.sup.5 and one in 10.sup.8 cells. Although this is a very rare event, it can have a large impact on the outcome of chemotherapy.
A tumor of average detectable size contains hundreds of millions of cells, some of which are likely to be drug resistant. Thus, although the mutations that produce drug resistance are quite rare, tumors containing some drug-resistant cells at the time of diagnosis may be the norm. One can easily predict the outcome of treating this type of tumor with a single drug. At first the patient will go into remission, wherein the tumor shrinks to an undetectable size because of the effects of the chemotherapy on the predominant drug-sensitive cells. However, the drug-resistant cells continue to multiply, to the point where they eventually dominate the cell population of the tumor. The tumor then grows to a size that results in the death of the patient. Experimental evidence has confirmed that even a single drug-resistant cell introduced into an otherwise curable tumor transplanted into a mouse will eventually multiply during the course of chemotherapy and dominate the tumor cell population, resulting in an incurable and ultimately fatal disease.
Theoretically it should be possible to solve this problem by administering a combination of drugs that act differently. This method of treatment is based upon the extremely small probability that two or more different drug resistances would arise spontaneously in the same cell. Combination chemotherapy appeared to obviate the problem of drug-resistant tumor cells.
Research was then directed to protocols for administering anticancer drugs in combinations. Newly developed drugs and combination chemotherapy several decades ago produced high cure rates for some childhood leukemias and for Hodgkin's disease. However, the major killers, such as lung cancer, breast cancer, and cancers of the gastrointestinal tract, remained resistant to chemotherapy.
The failures of combination chemotherapy were not understandable. Many theories were proposed to explain the observations, but few of these theories could be adequately tested. Early in the development of experimental chemotherapy in mice, simultaneous resistance of a number of drugs was an unexpectedly common occurrence. However, research focused on resistance to single agents, which at the time was more readily understood. In the late 1960's investigators experimented with drug-resistant tumor cells in vitro, and at that time the issue of multiple drug resistance resurfaced and some insights were gained into what is now known as the multidrug resistance phenotype.
These observations defined the properties of multidrug resistance. Although drug-resistant mutants were selected by means of a single anticancer drug, the cells were often simultaneously resistant (cross-resistant) to completely unrelated drugs. Most important was an observation arising from a number of indepcndent genetic experiments: multidrug resistance appeared to result from a single mutation, i.e., a single gene could account for the multiple cross-resistance to unrelated drugs.
This observation spurred research to find the multidrug-resistance gene in experimental tumors, stimulated inquiry into the gene's effect, and provided a rational explanation for the failures of combination chemotherapy. Since a single drug-resistant mutation is a rare event, the acquisition of multiple mutations in the same cell, yielding resistance to unrelated drugs, is an occurrence that is hugely improbable. The multidrug resistance phenotype that resulted from a single mutation explained how resistance to combination chemotherapy could be a common occurrence.
Working with various systems, investigators found that cells that were resistant to a drug somehow excluded the drug. There thus appeared to be some barrier that kept the drug from reaching the interior of the cell, where it would have its lethal effect. Two theories were provided to account for the evidence.
One theory proposed that a permeability barrier prevented drug entry into the cells. The other theory suggested that an efflux pump, a mechanism that actively pumped drug out of the cell once it had gotten inside, was at work in the resistant cells.
The latter model was based on the observations of the kinetics of drug flow into and out of the cells. It wa found that when a resistant cell was temporarily poisoned with cyanide to inhibit energy production, the cell behaved like a drug-sensitive one; it could not keep out the drug. When the cyanide was washed out and normal metabolism was restored, the cell could once again exclude the drug. Furthermore, the cell was then able to pump out the drug that had accumulated while it was poisoned. Thus, an energy-dependent drug-efflux pump seemed to be the simplest explanation.
Kartner et al., as reported in Scientific American, Mar., 1989, pp. 44-51, describe studies of Chinese hamster cells that were resistant to the drug colchicine. Components of the plasma membranes of the cells were separated by gel electrophoresis. This process revealed that there was a unique glycoprotein in the drug-resistant cells that appeared to be absent in the drug-sensitive cells. Glycoproteins are complex molecules made up of protein and carbohydrate, which are usually associated with the plasma membrane. The glycoprotein found associated with the Chinese hamster cells was rather large, having a molecular weight of approximately 170,000, and it was associated specifically with the plasma membrane. The glycoprotein was named P-glycoprotein for its association with the apparent permeability barrier to drugs that accompanied multidrug resistance.
A number of different groups reported similar findings with different tissue-culture systems. A variety of mouse, hamster, and human cells were selected for resistance to any one of a variety of known effective anticancer drugs: adriamycin, colchicine, daunomycin, vinblastine, vincristine, etc. All of these systems showed extensive cross-resistance to unrelated drugs, reduced intracellular accumulation of the drug involved, and alterations in the cell's surface membrane. The most consistent of the observed alterations was the appearance of a high-molecular-weight cell-surface glycoprotein similar in size to the P-glycoprotein.
It was later found that P-glycoprotein was a conserved molecule, i.e., a molecule that retains its structural identity across different mammalian species. Moreover, regardless of the species of origin or the drug of selection, the drug-resistant cells all exhibit a large elevation of P-glycoprotein expression in concert with the development of drug resistance.
Amino acid sequence studies and comparisons with other proteins led to a proposed model for P-glycoprotein structure which suggests possible ways that the protein provides multidrug resistance. It is likely that the 12 transmembrane regions of P-glycoprotein converge to form a 12-sided pore. On the outside of the cell there is little exposure of protein; this is the site where the sugar chains are attached. On the inside of the cell are two large, homologous domains projecting into the cytoplasm, which bear the ATP-binding sites. The sites that accept ATP on the P-glycoprotein molecule suggest that the protein has an energy-transducing function, such as the energy-dependent extrusion of toxic drugs from the cell.
It appears likely that the P-glycoprotein pumps drugs out of the cell in one of two ways. Either it binds a variety of drugs and extrudes them directly through the membrane by way of its putative transmembrane pore, or a second molecule (a carrier protein) binds to the drug and the drug-carrier complex is extruded across the membrane. There is evidence that some drugs may bind directly to P-glycoprotein, possibly as a first step in their ultimate transport across the surface membrane.
It has been established that in ovarian carcinomas, leukemia, and a variety of sarcomas, some of the tumors contain elevated levels of P-glycoprotein, In the small number of cases in which patient follow-ups have been possible, increased amounts of P-glycoprotein have been seen in combination with increasing unresponsiveness to chemotherapy. In perhaps 10 to 20 percent of the tumors tested, there has been clear evidence of elevated levels of P-glycoprotein.
Recently it has been found that a variety of compounds inhibit the pumping mechanism of P-glycoprotein, rendering multidrug-resistant tumor cells sensitive to drugs that would otherwise be ineffective. These compounds have been referred to as "chemosensitizers." Preliminary research indicates that some of these compounds act by interfering with the binding of drugs to P-glycoprotein, which appears to be a first step in its transport out of the cell.
There have been many reports in the prior art relating to the general concept of providing direct transport of an agent which is toxic to tumor cells directly to tumors. Reference is made, for example, to Hurwitz, E., "Attempts at Site Directed Experimental Chemotherapy with Antibody Drug-Conjugates", in Optimization of Drug Delivery, Alfred Benzon Symposium 17, Bundgaard, H. et al, ed., Munksgaard, Copenhagen 1982, pp. 253-269, and the various papers referenced on the first page thereof. Reports relating to the direct transport of a cytotoxic agent directly to tumors having .beta.-glucuronidase activity by conjugating the agent with glucuronic acid are discussed in detail in Rubin et al., U.S. Pat. No. 4,481,195, which patent is hereby incorporated by reference.
Rubin et al., in U.S. Pat. No. 4,481,195 and 4,337,760, disclose methods for treating hyperacidified tumors with .beta.-glucuronides. In the methods disclosed in these patents, tumor cells are selectively treated with nitrile-containing compounds with concurrent therapy to avoid the possibility of cyanide poisoning in the rest of the body.